Tag: p53

A team of Mount Sinai researchers have utilized induced pluripotent stem cells (iPSCs) to elucidate the genetic changes that seem to convert a well-known anti-cancer signaling gene into a driver bone cancers. When it comes to bone cancers, the survival rate has not improved in 40 years despite advances in treatment. Since this study might provide new targets and suggest new strategies for attacking such cancers. it represents a welcome addition to the cancer literature.

This study, which was published in the journal Cell, revolves around iPSCs, which were discovered in 2006 by Nobel laureate Shinya Yamanaka. iPSCs use genetic engineering and cell culture techniques to reprogram mature, adult cells to become like embryonic stem cells. These iPSCs are “pluripotent,” which means that they are able to differentiate into any adult cell type and can also divide in culture indefinitely.

For therapeutic purposes, iPSCs can be derived from a patient’s own cells, differentiated into the cells the patient needs to be replaced, and then implanted into the patient’s body to augment tissue healing or even organ reconstruction. Since iPSCs can be successfully differentiated into heart muscle, nerve cells, bone, and other cell types, they have the potential advance the field of regenerative medicine by leaps and bounds.

iPSCs have already made their presence known in the clinic by serving as model systems for research and diagnosis. The new Mount Sinai study used iPSCs to construct an accurate model of a genetic disease “in a dish.” The culture dishes contain self-renewing patient-specific iPSCs or a specific cell line that enable in-depth study diseases that are driven by each person’s genetic differences. When matched with patient records, iPSCs and iPSC-derived target cells have the ability to help physicians predict a patient’s prognosis and whether or not a given drug will be effective for him or her.

In this study, skin cells from healthy patients and patients with a genetic disease called Li-Fraumeni syndrome were isolated and reprogrammed into patient-specific iPSC lines. These iPSCs were then differentiated into bone-making cells (osteoblasts), which are the cells where particular rare and common bone cancers start. Li-Fraumeni syndrome greatly predisposes patients to a variety of cancers in several different types of tissues.

The patient-derived osteoblasts were then tested for their tendencies to become tumor cells and to make bone. This particular bone cancer model did a better job of recapitulating the characteristics of bone cancer than previously used mouse or cellular models.

“Our study is among the first to use induced pluripotent stem cells as the foundation of a model for cancer,” said lead author Dung-Fang Lee, PhD, a postdoctoral fellow in the Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai. “This model, when combined with a rare genetic disease, revealed for the first time how a protein known to prevent tumor growth in most cases, p53, may instead drive bone cancer when genetic changes cause too much of it to be made in the wrong place.”

The Mount Sinai disease model research uses a simple fact of human life as its basis: human genes undergo mutations at a certain rate that tends to increase as we age, and the formation of new mutations in relentless and constant. Some mutations make no difference, a few some confer advantages, and others cause disease. Beyond inherited mutations that contribute to cancer risk, the combination of random, accumulated DNA changes in our cells as we age also increase our cancer risk.

The current study focused on those genetic pathways involved in Li-Fraumeni Syndrome or LFS, a rare genetic disease that causes high risk for many cancers in affected families. Osteosarcoma (bone cancer) is a common cancer observed in LFS patients and many of them are diagnosed before the age of 30. Additionally, osteosarcoma is the most common type of bone cancer in all children, and after leukemia, the second leading cause of cancer death for them.

Importantly, about 70 percent of LFS families have a mutation in their copy of a genes called TP53, which encodes the p53 protein. P53 is a “the tumor suppressor,” which means that it functions to preserve the integrity of the genome and keep the rate of cell division in check. Common forms of osteosarcoma, which are driven by somatic or inherited mutations, have also been closely linked by past studies to defects in p53 when mutations interfere with the ability of the protein to function properly.

Crystal Structure of p53 protein bound to DNA

Rare genetic diseases like LFS provide excellent model systems because they tend to result from a change in a single gene, instead of the diverse and overlapping mutations observed in common diseases, and, in this case, more common, non-inherited bone cancers. The LFS-iPSC based modeling highlights the contribution of p53 alone to osteosarcoma.

By analyzing iPSC lines, and bone cancer driven by p53 mutations in LFS patients, the Munt Sinai research team showed, for the first time, that the LFS bone cancer results from an overactive p53 gene. Too much p53 in osteoblasts dampens the function of a gene, H19, and a related protein, decorin, that would otherwise help stem cells differentiate into normal osteoblasts.

The inability of cells to differentiate makes them vulnerable to genetic mistakes that drive cancer, since more “stemness” means a tendency toward rapid, abnormal growth, like that observed in tumors. One tragic feature of osteosarcoma is the rapid, error-prone production of weaker bone by cancerous bone-making cells, where a young person surprisingly breaks a bone to reveal undiagnosed, advanced cancer.

Dung-Fang Lee and his colleagues discovered that the H19 gene seems to control a network of interconnected genes that fine-tune the balance between cell growth and resistance to growth. Decorin is a protein that is part of connective tissue like bone, but that also plays a signaling role, interacting with growth factors to slow the rate that cells divide and multiply, unless turned off by too much p53.

“Our experiments showed that restoring H19 expression hindered by too much p53 restored “protective differentiation” of osteoblasts to counter events of tumor growth early on in bone cancer,” said co-author, Ihor Lemischka, PhD, Director of The Black Family Stem Cell Institute within the Icahn School of Medicine. “The work has implications for the future treatment or prevention of LFS-associated osteosarcoma, and possibly for all forms of bone cancer driven by p53 mutations, with H19 and p53 established now as potential targets for future drugs.”

In order to make induce pluripotent stem cells, scientists need to reprogram existing cells to form a cell that is undifferentiated and ready to become whatever we want it to become. Reprogramming is achieved by increasing the concentration of four different proteins within the cells. To increase the intracellular concentration of these proteins, scientists use viruses or other vehicles to import the genes that encode these proteins into the cells and the increased production of these proteins drives cells to become embryonic-like cells that can become anything we want them to be. Unfortunately, reprogramming, at present, is rather inefficient.

Recently, work from five different labs has shown that two biochemical pathways, the p16INK4A and ARF–p53 pathways, put the brakes on iPSC formation. Five papers in a recent edition of the journal Nature show that the components of this pathway are silenced in iPSCs and strongly expressed in terminally differentiated cells. There are three genes found at the site known as Ink4/Arf (p16Ink4a, p19Arf and p15Ink4b), and these genes are absent in several different types of cancers. For example p16Ink4a is inactive in about 90% of all pancreatic cancers. p15Ink4b is absent from several different blood-based cancers and loss of p19Arf is involved in melanoma formation. Each one of these gene products acts as a barrier to reprogramming and iPSC formation.

p16INK4A and p19ARF positively regulate the p53 pathway. p53 inhibits cell proliferation and promotes cellular senescence. During senescence, the cell essentially takes a nap. If a you want a cell to grow and participate in healing, the induction of senescence is not a good thing, but if a cell is growing uncontrollably and contributing to a tumor, then forcing a cell into senescence is a good thing. A protein called p21 (CDKN1A) is also upregulated by p53, and this protein inhibits proliferation and promotes senescence. Thus p53 is one of the major switches that prevents cell proliferation and promotes senescence. In 2008, Zhao and coworkers showed that interfering with p53 activity promoted iPSC formation (Zhao, et al., Cell Stem Cell 3, 475-79, 2008).

Li et al. and Utikal et al. observed that the INK4/ARF locus is silenced in iPS cells reprogrammed from Mouse Embryonic Fibroblasts (MEFs), as well as in embryonic stem cells, but not in MEFs (Li, H. et al. Nature460, 1136–1139 (2009) & Utikal, J. et al. Nature460, 1145–1148 (2009)). Utikal et al. also observed that older MEFs, which harbor increased levels of p16INK4A, ARF and p21 owing to ageing and the onset of senescence, show a decrease in reprogramming efficiency. Li et al. also linked ageing and expression from the INK4/ARF locus with decreased iPS cell generation. They showed that cells from old mice express genes at this locus at a higher level than cells from young mice and that this is associated with a decreased reprogramming efficiency, which can be rescued by knocking down INK4/ARF.

As a final caveat, Marión et al. found that p53 prevents the reprogramming of MEFs that have various types of DNA damage (Marión, R. M. et al. Nature460, 1149–1153 (2009)). Although loss of p53 function allows faster and more efficient reprogramming in the presence of DNA damage, it generates iPS cells that contain damaged DNA and chromosomal abnormalities. This emphasizes that, although these studies provide crucial mechanistic insight into how the generation of iPS cells is regulated, it will be important to determine how the p16INK4A and ARF–p53 tumor suppressor pathways can be silenced to allow the efficient production of iPS cells without increasing the possibility of making cell lines that contain mutations that predispose them to malignant tumor formation.

Surely iPSC production represents the future of regenerative medicine. We need not kill human embryos, and the time required to make iPSCs can be substantially cut with this technology as it is honed and even made safer.